TFIID dependency of steady-state mRNA transcription altered epigenetically by simultaneous functional loss of Taf1 and Spt3 is Hsp104-dependent

In Saccharomyces cerevisiae, class II gene promoters have been divided into two subclasses, TFIID- and SAGA-dominated promoters or TFIID-dependent and coactivator-redundant promoters, depending on the experimental methods used to measure mRNA levels. A prior study demonstrated that Spt3, a TBP-delivering subunit of SAGA, functionally regulates the PGK1 promoter via two mechanisms: by stimulating TATA box-dependent transcriptional activity and conferring Taf1/TFIID independence. However, only the former could be restored by plasmid-borne SPT3. In the present study, we sought to determine why ectopically expressed SPT3 is unable to restore Taf1/TFIID independence to the PGK1 promoter, identifying that this function was dependent on the construction protocol for the SPT3 taf1 strain. Specifically, simultaneous functional loss of Spt3 and Taf1 during strain construction was a prerequisite to render the PGK1 promoter Taf1/TFIID-dependent in this strain. Intriguingly, genetic approaches revealed that an as-yet unidentified trans-acting factor reprogrammed the transcriptional mode of the PGK1 promoter from the Taf1/TFIID-independent state to the Taf1/TFIID-dependent state. This factor was generated in the haploid SPT3 taf1 strain in an Hsp104-dependent manner and inherited meiotically in a non-Mendelian fashion. Furthermore, RNA-seq analyses demonstrated that this factor likely affects the transcription mode of not only the PGK1 promoter, but also of many other class II gene promoters. Collectively, these findings suggest that a prion or biomolecular condensate is generated in a Hsp104-dependent manner upon simultaneous functional loss of TFIID and SAGA, and could alter the roles of these transcription complexes on a wide variety of class II gene promoters without altering their primary sequences. Therefore, these findings could provide the first evidence that TFIID dependence of class II gene transcription can be altered epigenetically, at least in Saccharomyces cerevisiae.


Introduction
To initiate transcription in eukaryotes, TATA-binding protein (TBP) first binds to the TATA box or TATA-like element [1], and subsequently recruits other general transcription factors, Mediator and RNA polymerase II, to assemble the preinitiation complex (PIC) at the core promoter region [2][3][4][5]. TBP is an integral subunit of TFIID and is generally delivered to the promoter of class II genes via TFIID or SAGA, which are related multi-protein complexes containing five Tafs as common components [3,[6][7][8].
Class II genes can be divided into two subclasses, TFIID-or SAGA-dominated genes, depending on whether steady-state mRNA levels are impaired more robustly by TFIID or SAGA mutations, respectively [9]. A recent study potentially challenged this dogma, demonstrating that nascent mRNA levels of most (~87%) analyzable class II genes are significantly affected by depletion of TFIID, but not of SAGA, while the remainder of class II genes (~13%) are affected modestly by single depletion of either TFIID or SAGA, but significantly affected by depletion of both TFIID and SAGA [10]. These newly defined types of class II genes are designated as "TFIID-dependent" and "coactivator-redundant (CR)," respectively [10]. TBP is delivered to the TATA element of TFIID-dependent genes solely via TFIID, while either TFIID or SAGA facilitates delivery of TBP to CR genes [10]. Notably, TFIID-dependent and CR genes overlap with, but are not identical to, TFIID-and SAGA-dominated genes, respectively. This seeming discrepancy could be attributed to the experimental methods exploited in these two studies [9,10], which either measured nascent [10] or steady-state [9] mRNA levels for classification. Although it is certain that at least two distinct types of class II gene promoters exist, the distinct roles of TFIID and SAGA in transcription from class II promoters remain incompletely understood.
To facilitate transcription, activators that bind DNA at a distance from their target promoters make physical contact with the general transcription machinery assembled near the transcription start site by looping out the intervening DNA [11,12]. During this event, the entire transcriptional machinery, including activators, coactivators, RNA polymerase II, and even the transcribed RNA, could form large separated assemblies in a liquid-like state, referred to as transcriptional condensates, primarily via weak multivalent interactions among intrinsically disordered regions (IDRs) of these components [13][14][15][16][17]. These condensates enable efficient transcription by increasing localized concentrations of transcription factors and DNA. In living cells, activators bind to only a limited number of the genomic sites that contain their cognate DNA recognition motifs. In this context, it is notable that the long IDRs of the two distinct types of yeast activators, Msn2 and Yap1, play a crucial role in achieving this selective binding in vivo [18]. Thus, transcriptional condensates could also provide a means for activators to efficiently locate their target sites, for example by enabling a facilitated diffusion search within a restricted zone of the nucleus [19]. Furthermore, a recent study revealed that Taf14, a histone reader YEATS family protein that physically associates with not only TFIID but also with several other transcription complexes, undergoes liquid-liquid phase separation in vitro [20]. This suggests that Taf14 could serve as a central hub in formation of some transcriptional condensates. Despite these recent findings, it is currently unknown whether the two types of class II gene promoters regulated differentially by TFIID and SAGA can form distinct promoter type-specific transcriptional condensates that are differentially dependent on TFIID and/or SAGA in transcription. PGK1 encodes phosphoglycerate kinase, an enzyme that catalyzes an ATP-generating step of glycolysis, and its transcription is driven by a very strong TATA box-containing promoter [21][22][23]. This promoter was assigned as the "SAGA-dominated" [9] and "CR" [10] types, and thus its transcription is Taf1/TFIID-independent, when measured by steady-state mRNA levels [24][25][26][27]. In a previous study, we reported that SPT3, which encodes a TBP-regulatory SAGA subunit, facilitates PGK1 transcription in a TATA box-dependent manner [25]. Interestingly, introduction of plasmid-borne SPT3 into spt3Δ or spt3Δ taf1 mutant strains rescued TATA box-dependent PGK1 transcription but not Taf1/TFIID independence, particularly in transcription from a PGK1 promoter lacking a TATA box [25]. However, the regulatory mechanism for this phenomenon, in which plasmid-borne SPT3 failed to restore Taf1/TFIID independence, has yet to be identified.
In the present study, we first determined whether the gene location of SPT3, for example chromosomal or plasmid-borne, affected Taf1/TFIID-independent PGK1 transcription, revealing that Taf1/TFIID dependence could be acquired regardless of gene location when Spt3 and Taf1 functions were compromised simultaneously during strain construction. Thus, the transcriptional mode of the PGK1 promoter could be switched epigenetically from Taf1/ TFIID-independent to Taf1/TFIID-dependent without altering the final yeast strain genotype. This was an unexpected finding, as most prior studies suggest that TFIID dependence or independence is determined by the promoter structure, including the upstream activating sequence (UAS), core promoter element, or their combination [24,[28][29][30][31][32][33]. Further analyses demonstrated that potential prion-like factor(s) generated in an Hsp104-dependent manner during strain construction affected the transcription mode not only of the PGK1 promoter, but also of many other class II gene promoters. Based on these new findings, we suggest the intriguing possibility that TFIID and SAGA could form transcriptional condensates that support efficient transcription of a range of class II genes, and thereby that simultaneous functional loss of these two factors could alter the original traits of these condensates, switching the transcriptional mode from Taf1/TFIID-independent to Taf1/TFIID-dependent, even for the same promoter, without changing its primary structure.

Yeast strains
Standard techniques were used for yeast growth and transformation [34]. Yeast strains used in the study are listed in S1 Table. Oligonucleotide sequences used for strain or plasmid construction are listed in S2 Table. All strains used in the study were generated from BY4741 or BY4742, as described below.

Northern blot analysis
Northern analysis was performed as described previously [25]. For detection of PGK1, VTC1, and SCR1, DNA fragments were amplified by PCR from genomic DNA of BY4741, purified, and 32 P-labeled by random priming with the Klenow fragment. The PCR primer pairs used were as described previously [25].

Reverse transcription-quantitative PCR (RT-qPCR) analysis
Total RNA was isolated from the indicated strains grown to log-phase at 25ºC in synthetic complete media or further incubated at 37ºC for 2 h, as described previously [25]. cDNA synthesis was performed on 2 μg total RNA using random hexamers (Invitrogen) and Rever Tra Ace (Toyobo) according to the manufacturer's protocol. RT-qPCR was performed on 1:5 diluted cDNA using a Light Cycler 96 system (Roche) with 1x THUNDERBIRD qPCR Mix (Toyobo) in a total reaction volume of 10 μL containing 0.3 μM of the appropriate primer pair. The results were analyzed using Light Cycler 96 Application Software Version 1.1 (Roche).

RNA-seq analysis
tocol #2] and YTK20049 [protocol #3]) strains cultured in the presence or absence of 3 mM Gdn-HCl were examined using RNA-seq analysis as described previously, with minor modifications [45]. Briefly, total RNA from each strain was prepared using ISOGEN with a Spin Column (Nippon Gene, Tokyo, Japan). cDNA sequencing libraries were generated from 1000 ng total RNA using a NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer's protocol. The libraries were amplified with seven PCR cycles. Pooled libraries were sequenced on an Illumina NextSeq 500 instrument, generating 75-bp single-end reads with 6-bp index tags. Analyses of sequenced data were conducted using CLC Genomics Workbench (QIAGEN) ver. 20.0.4. The single-end reads were trimmed to a fixed length of 35-bp from the 3'-end using the Trim Reads tool. Trimmed reads were mapped to the S. cerevisiae s288c reference genome (NC_001133-NC_001148) using the RNA-seq tool, with default parameters. Read data for RNA-seq experiments are accessible on DDBJ Sequence Read Archive under accession no. DRR403518-DRR403553. Clustering, volcano plot, and Venn diagram analyses were performed using in-house Python script.

Taf1/TFIID dependence of PGK1 transcription depends on the construction protocol of the SPT3 taf1-N568Δ strain
A prior study demonstrated that Spt3 has two distinct functions on the PGK1 promoter, TATA box-dependent 2-3-fold stimulation and conferring Taf1/TFIID independence of PGK1 transcription [25]. Intriguingly, plasmid-borne non-tagged SPT3 restored TATA boxdependent PGK1 transcription, but not Taf1/TFIID-independent PGK1 transcription, especially when the promoter TATA box was mutated to the GAGA sequence in the spt3Δ taf1-N568Δ strain [25]. Because SPT3 expression driven by both low-copy and high-copy plasmids yielded similar results, specific loss of Taf1/TFIID-independent PGK1 transcription was likely due to differences between chromosomal and plasmid-borne SPT3 rather than simply to differences in Spt3 expression levels. Of note, Taf1/TFIID dependence of PGK1 transcription was examined by the experimental procedure using a temperature shift from 25˚C to 37˚C, as the taf1 strains used in our studies carried a temperature-sensitive taf1-N568Δ allele.
To further explore the regulatory mechanism of this phenomenon, we employed the same VTC1 reporter system used in a previous study [25], but quantified mRNA levels by RT-qPCR instead of Northern blot analysis. Using this reporter system, in which the endogenous VTC1 promoter was replaced with test promoters, the transcriptional activities not only of modified or unmodified PGK1 promoters, but also the transcriptional activity of the endogenous PGK1 promoter, could be measured simultaneously in the same strain. Consistent with prior findings [25], plasmid-borne V5 epitope-tagged SPT3 expressed from a high-copy plasmid failed to confer Taf1/TFIID independence to the PGK1 promoter when the TATA box was mutated to GAGA (Fig 1A and 1B, lanes 6 [VTC1]. Note that the gene name in square parenthesis Strains were cultivated at 25ºC and further incubated at 37ºC for 2 h in synthetic medium containing 2% glucose, as indicated at the right side of the VTC1 panel. mRNA levels (mean ± SD of three biological replicates) were quantified and normalized to SCR1 signal. All data are presented relative to that of lane 1 (25˚C). The significance of differences between cultivation at 25˚C and 37˚C was assessed for each strain by t-test: � p < 0.05; �� p < 0.01. Reporter VTC1 mRNA was more heat-sensitive than endogenous PGK1 mRNA, even in the wild type strain (lane 1), as already shown by Northern blot analyses in a previous study [25]. The results from spt3Δ cells are shaded (lanes 3-4 and 7-8), while those obtained from SPT3 taf1 cells are labeled with gray bars at the top of the graph (lanes 2 and 6). (B) RT-qPCR analyses to measure mRNA levels of PGK1 (top panel) or VTC1 (bottom panel) in the eight strains carrying the VTC1 reporter driven by the PGK1 promoter, in which the TATA box was intact (lanes 1-4) or substituted with the GAGA sequence (lanes 5-8), as indicated below the bottom panel. In these strains, chromosomal TAF1 was deleted and substituted with plasmid-following lane number hereafter indicates the mRNA that is referred, for example [PGK1, VTC1] in the following sentence. Notably, PGK1 transcription was also Taf1/TFIID-dependent with plasmid-borne SPT3, albeit weakly, even when the TATA box was intact (Fig 1A and 1B Next, we determined if chromosomal V5 epitope-tagged SPT3 could confer Taf1/TFIID dependence to the PGK1 promoter, similar to plasmid-borne SPT3, when expressed at severalfold higher levels by the ectopic TDH3 promoter. Chromosomal SPT3 did not confer Taf1/ TFIID dependence to the PGK1 promoter, regardless of whether it was driven by the endogenous SPT3 promoter (Fig 1C, lanes 2  Simultaneous functional loss of Spt3 and Taf1 during strain construction renders PGK1 transcription Taf1/TFIID-dependent in the SPT3 taf1-N568Δ strain As described above, the SPT3 taf1-N568Δ strain, generated from the spt3Δ taf1-N568Δ strain by reintroduction of SPT3 via either plasmid transformation [25] (Fig 1B) or chromosomal integration (Fig 1C, third and fourth panels), demonstrated that PGK1 transcription was Taf1/ TFIID-dependent, while PGK1 transcription remained Taf1/TFIID-independent in the SPT3 taf1-N568Δ strain generated from a distinct SPT3 taf1-N568Δ strain by directly replacing SPT3 via genomic recombination (Fig 1C, first and second panels). These observations suggest that Taf1/TFIID dependence could be conferred to the PGK1 promoter in the SPT3 taf1-N568Δ strain only when Spt3 and Taf1 functions were compromised simultaneously during strain construction.
Consistent with above finding, RT-qPCR revealed that PGK1 transcription was Taf1/ TFIID-dependent only in the SPT3 taf1-N568Δ strain generated by protocol #3 (S1B Fig, lanes  9 and 10 [PGK1, VTC1]). This supports the conclusion that simultaneous functional loss of Spt3 and Taf1 during strain construction is a prerequisite for rendering PGK1 transcription Taf1/TFIID-dependent in the SPT3 taf1-N568Δ strain.
Trans-acting promoter reprogramming factor(s) are produced in the SPT3 taf1-N568Δ strain experienced with simultaneous functional loss of Spt3 and Taf1 during strain construction The observation that PGK1 transcription was Taf1/TFIID-dependent in the SPT3 taf1-N568Δ strain specifically when it was generated by protocol #3 (S1 Fig), in which both Spt3 and Taf1 function were lost simultaneously during strain construction, raised the intriguing possibility that as-yet unidentified factors produced only under simultaneous loss of Taf1 and Spt3 could reprogram the transcription mode of the PGK1 promoter from a Taf1/TFIID-independent state to a Taf1/TFIID-dependent state. To begin to evaluate this possibility, we sought to identify whether such promoter reprogramming factor(s) functioned in cis in a manner tightly associated with the originally reprogrammed PGK1 promoter, or rather in trans in a more diffusible manner, for example even if the PGK1 promoter was later introduced into cells.
We thus generated a VTC1 # allele carrying eight silent point mutations in the middle of the open reading frame (ORF) (Fig 2A), such that the mRNA transcript of the VTC1 # allele could be distinguished from that of the original VTC1 allele by RT-qPCR, as evidenced by the observation that specific amplification signals were not detected by quantitation software for cDNA samples derived from the VTC1 strain. Specificity of the primer pairs used for RT-qPCR was also confirmed by conventional RT-PCR (S2 Fig). The SPT3 taf1-N568Δ strains harboring either VTC1 or VTC1# were generated by protocol #2 or #3 and PGK1 transcription was measured ( Fig 2B). PGK1 transcription was Taf1/TFIID-dependent in SPT3 taf1-N568Δ strains generated by protocol #3 (Fig 2B, lanes 5, 6, 11, and 12 [PGK1, VTC1, VTC1 # ]), but not in strains generated by protocol #2 (Fig 2B, lanes 3, 4, 9, and 10 [PGK1, VTC1, VTC1 # ]), regardless of which reporter alleles they harbored.

PLOS ONE
TFIID-dependency of mRNA transcription can be altered epigenetically

Potential prion-like properties of promoter reprogramming factor(s)
The reprogramming phenomenon observed for the PGK1 promoter was likely to be an epigenetic event, as all the tested SPT3 taf1-N568Δ strains carried essentially the same genetic backgrounds, irrespective of whether PGK1 transcription was Taf1/TFIID-independent or Taf1/ TFIID-dependent. Thus, we subsequently examined the inheritance of this unexpectedly acquired trait from mother diploid strains (Fig 2C, lanes 5/9 and 6/10) to haploid progenitors (S3 Fig). This trait was inherited by progenitors evenly but somewhat weakly, as exemplified more evidently by the results obtained for progenitors carrying the VTC1 # reporter gene driven by the GAGA-substituted PGK1 promoter (compare lanes 21-32 with lanes 4 and 8 in S3 Fig). This non-mendelian inheritance strongly supports the notion that this alteration of transcription mode was an epigenetic event, involving trans-acting and meiotically unstable promoter reprogramming factor(s), to render the PGK1 promoter Taf1/TFIID-dependent.

Hsp104 plays an essential role in producing promoter reprogramming factor(s)
Because we identified that Gdn-HCl inhibition of Hsp104 affected Taf1/TFIID dependence of the PGK1 promoter, we next sought to examine more directly the effect of Hsp104 by genetic approaches utilizing the hsp104Δ mutation. For this purpose, test haploid strains were constructed by protocol #2 or #3 after disruption of the HSP104 gene (Fig 3A). We did not detect significant differences in the PGK1 transcription profiles between these two types of strains, regardless of whether they were constructed by protocol #2 or #3 (Fig 3B, lanes 4-7 [VTC1, PGK1] and 11-14 [VTC1 # , PGK1]), indicating that Taf1/TFIID dependence could be conferred to the PGK1 promoter specifically by protocol #3 only when Hsp104 function was intact. Because the hsp104Δ mutation did not affect PGK1 transcription (compare the results of Figs 2B and 3B, based on those of lanes 1 obtained for the same strain), Hsp104 likely had an essential role in production of promoter reprogramming factor(s) but not for  1-7) or the VTC1 # reporter (lanes [8][9][10][11][12][13][14] driven by the PGK1 promoter in which the TATA box was intact (lanes 1, 2, 4, 6, 8, 9, 11, and 13)

PLOS ONE
TFIID-dependency of mRNA transcription can be altered epigenetically transcriptional regulation itself. Northern blot analyses using probes targeted for different regions of PGK1 and VTC1 ORFs from those amplified by the RT-qPCR primers (S5 Fig) were consistent with RT-qPCR findings, minimizing the possibility that the findings were due to unexpected artifacts such as transcript sequence variance.

Genome-wide RNA-seq analyses of strains constructed by protocol #2 or #3 in the presence or absence of Hsp104 inhibition or mutation
The above analyses indicated that promoter reprogramming factor(s) that switch the transcription mode of the PGK1 promoter could be produced in SPT3 taf1-N568Δ strains constructed by protocol #3 in an Hsp104-dependent manner. To examine how broadly these factor(s) could reprogram the transcription mode of other class II genes as well as how extensively this reprogramming could be affected by Gdn-HCl treatment or the hsp104Δ mutation, we conducted genome-wide RNA-seq analyses for the SPT3 TAF1 (wild type [WT] as a control) and SPT3 taf1-N568Δ strains constructed by protocol #2 or #3 (hereafter designated as #2 strains or #3 strains) in the presence or absence of Gdn-HCl treatment or the hsp104Δ mutation.
As expected, volcano plot analyses of TPM (transcripts per million) data revealed that the gene expression profiles of the #2 and #3 strains were significantly different from that of the WT strain at 37˚C (Fig 4A and 4B) and even at 25˚C (S6A and S6B Fig). Importantly, the gene expression profiles of the #3 strains were significantly different from those of the #2 strains, regardless of whether the strains were incubated at 37˚C (Fig 4C and 4F) or 25˚C (S6C and S6F Fig) in the absence (Fig 4C, S6C Fig) or presence (Fig 4F, S6F Fig) of Gdn-HCl. In greater contrast, we found that the gene expression profiles of the entire set of genes were nearly the same for the #2 and #3 strains under the presence of the hsp104Δ mutation when they were incubated at 37˚C (Fig 4I) or 25˚C (S6I Fig). These observations suggest that promoter reprogramming factor(s), which could be produced specifically in the #3 strains in an Hsp104-dependent manner, affect the transcription mode not only of the PGK1 promoter but also of many other class II gene promoters.
Two distinct types of genes in budding yeast were recently distinguished, which are designated as "coactivator-redundant (CR)" and "TFIID-dependent" genes [10]. Transcription of CR genes is modestly affected by rapid depletion of either TFIID or SAGA singly, but is severely affected by simultaneous depletion of TFIID and SAGA. On the other hand, transcription of TFIID-dependent genes is severely affected by rapid depletion of TFIID alone, and is only modestly affected by SAGA depletion [10]. Because PGK1 is a CR gene [10], we examined the possibility that differences of gene expression levels between #2 strains and #3 strains could be more significant for CR genes than for TFIID-dependent genes. To test this hypothesis, the numbers of affected genes (criteria: > 2-fold change, p < 0.05) were counted not only for the entire set of genes, but also for the two types of genes separately (Fig 4A-4I, S6A-S6I Fig). The lists of these counted genes are summarized in S3 Table (37˚C) and 4 (25˚C). Among the total 655 CR genes, the percentages of differentially expressed CR genes between #2 strains and #3 strains were greater than that of similarly affected differentially expressed (> 2-fold change, p < 0.05) TFIID-dependent genes (4245 genes total), regardless of incubation temperature or Gdn-HCl treatment (Fig 4C and 4F and S6C and S6F Fig). Thus, CR genes were likely more sensitive to the effects of the promoter reprogramming factor(s) than TFIID-dependent genes.
Further, Venn diagrams revealed that the differentially expressed genes between WT and #2 strains (Fig 4A, 4D and 4G) were not as significantly affected by Gdn-HCl treatment or hsp104Δ mutation, as the differentially expressed genes between WT and #3 strains (Fig 4B, 4E and 4H), at least when the strains were incubated at 37˚C (Fig 4J and 4K). This indicated that Volcano plot analyses of the gene expression profiles of #2 and #3 strains reveal HSP104 dependency of differential gene expression. TPM data of the RNA-seq analyses obtained from two biological replicates of six strains with and without taf1 or hsp104Δ mutations after cultivation under various conditions, which are indicated in the bottom panel, were averaged and subjected to volcano plot analyses. Fold changes of gene expression in the #2 strain or #3 strain relative to that in the TAF1 strain are shown in A and B, respectively. Similarly, fold changes of gene expression in the #3 strain relative to that in the #2 strain are shown in C. The analogous three sets of comparisons were conducted for the TPM data derived from strains cultivated in the presence of Gdn-HCl (D, E, F) or the hsp104Δ mutation (G, H, I). In A-I, the genes considered to be significantly differentially expressed (> 2-fold change, p < 0.05) are indicated with red dots, while ribosomal protein genes,~95% of which are TFIID-dependent genes [10], are indicated with green dots. Furthermore, the numbers of class II genes or those of the subclasses CR or TFIIDdependent, that were significantly differentially expressed, are shown in the upper left or upper right corners of A-I. The overlaps among these differentially expressed class II genes across A, D, and G (#2 strain versus TAF1) Gdn-HCl treatment could alter the properties of promoter reprogramming factor(s) in a manner different from that of the hsp104Δ mutation, which is consistent with its complex effects on the PGK1 promoter ( S4 Fig). Finally, hierarchical clustering analysis for the same TPM data (S7 Fig) revealed that the PGK1 promoter driving expression of VTC1 reporter gene as well as endogenous PGK1 was grouped into a subcluster comprising the 21 most highly expressed genes (S8A Fig), which was visualized at the top of the heatmap (S7 Fig). Intriguingly, 12 out of the 21 most highly expressed genes were related to the processes of either glycolysis or ethanol fermentation (S8B Fig), suggesting that promoters of glycolytic and fermentative genes could be regulated by a common mechanism that was similarly affected by several distinct genetic alterations (taf1, spt3Δ, hsp104Δ), by the order of mutation introduction during strain construction and/or physicochemical perturbations such as Gdn-HCl or increased temperature.

Discussion
In the present study, we demonstrated that the transcription mode of the PGK1 promoter could be switched from TFIID-independent to TFIID-dependent by as-yet unidentified promoter reprograming factor(s) produced specifically in yeast strains that underwent simultaneous functional loss of Taf1 and Spt3 during their construction. Intriguingly, production of these factor(s) was Hsp104-dependent, and affected transcription not only of the PGK1 promoter but also those of many other class II genes, including coactivator-redundant (CR) and TFIID-dependent genes.
Hsp104 plays an essential role in propagation of the amyloid fiber-forming type of prions by several distinct mechanisms, including increased number of prion seeds due to its severing activity [48]. On the other hand, a recently identified non-amyloid type of prion, which is primarily comprised of nucleic acid binding proteins with large intrinsically disordered domains (IDDs), can be propagated via Hsp104, Hsp70, Hsp90, or other chaperones [49]. The present observation that the promoter reprograming trait was genetically dominant and inherited to progenitors in a non-mendelian fashion implies that the promoter reprogramming factor(s) could potentially be either a type of prion, albeit with some unconventional properties such as meiotic instability and a complex response to Gdn-HCl treatment.
Recent studies have demonstrated that many components of the transcription machinery, including the sequence-specific transcriptional regulators, Med1, p300, Brd4, P-TEFb, and RNA polymerase II, can form transcriptional condensates individually or collectively, which are membrane-less compartments built up with RNA and/or DNA in the nucleus to support efficient transcription via a liquid-liquid phase separation mechanism [17,[50][51][52]. Because cytoplasmic biomolecular condensates such as stress granules [53] or heat-inducible condensates [54] require Hsp104 for their dynamic behaviors, the phase transition of some nuclear transcriptional condensates could also be mediated by these chaperone functions. If so, another possibility is that the promoter reprogramming factor(s) might not be a prion form of particular protein(s), but rather reflect a certain phase-transitioned state of the transcriptional machinery that is induced by the simultaneous functional loss of Taf1 and Spt3 during strain construction. To further explore these possibilities, identification of causal or involved protein (s) as well as examination of their physical properties would be required, especially considering that proteins such as Sup35 [55] and p53 [56] form not only amyloid fibrils but also biomolecular condensates.
One of the most remarkable observations in the study was that the genome-wide gene expression profile of the #3 strain was very similar to that of the #2 strain under the presence of the hsp104Δ mutation when cultured at either 37˚C (Fig 4I) or 25˚C (S6I Fig). On the other hand, the genome-wide gene expression profile of the #2 strain was significantly altered by the hsp104Δ mutation at both temperatures (Fig 4J and S6J Fig), indicating that Hsp104 plays a crucial role not only in generating promoter reprograming factor(s) but also in maintaining the original gene expression profile of the #2 strain. Furthermore, it should be noted that the difference of the gene expression profile between the WT and #2 strains was significantly diminished by the presence of the hsp104Δ mutation in culture at 25˚C (S6G Fig). This implies that Hsp104 could allow #2 strain to acquire a unique gene expression profile under permissive growth conditions, presumably via the aid of either prion(s) or condensate(s) that would be generated ectopically in the #2 strain, even in which the functional losses of TFIID and SAGA were minimal and null, respectively. Importantly, the identity of the factors generated in the #2 strain and their relationship to the promoter reprograming factor(s) generated in the #3 strain are unclear. In any case, these observations raised an intriguing possibility that the altered gene expression profiles obtained at restrictive temperatures for the taf1-N568Δ mutant (#2 strain) as well as for many other previously reported taf mutants [24,[57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72] could be achieved by the combined effects not only of impaired TFIID function but also of other prion (s) or condensate(s) ectopically generated in taf mutant strains.
TFIID and SAGA both play important roles in transcription of nearly all class II genes [10,73,74]. Prior studies suggest that TBP loading for TFIID-dependent genes is mediated by TFIID, while TBP loading for CR genes is mediated redundantly by TFIID and SAGA [10]. Furthermore, chromatin modifications established by SAGA and eliminated slowly after SAGA depletion are important for transcription of both TFIID-dependent and CR genes [10]. These views were obtained by the studies using ChEC-seq and/or nascent transcript labeling techniques instead of more traditional ChIP-seq and/or steady-state transcript analyses. The amounts of steady-state mRNAs do not accurately reflect the activity of ongoing transcription, especially in yeast cells defective of pol II transcriptional machinery, including TFIID and SAGA, as these transcriptional defects are often masked by cytoplasmic stabilization of the corresponding mRNAs [73][74][75][76][77]. This "transcript buffering" effect [77] could explain the apparently limited effects of some TFIID and SAGA mutations on global transcription assessed by measurement of steady-state mRNAs. Therefore, the transcription mode of some genes could be revealed as being altered from TFIID-independent to TFIID-dependent, similar to that of the PGK1 promoter, if the transcript buffering system becomes dysfunctional and thereby unable to compensate for impaired TFIID activity. Considering that the Ccr4-Not complex plays a crucial role in cytoplasmic transcript buffering [77] and interacts genetically with TFIID and SAGA [78,79], this is a potential candidate for the aforementioned promoter reprograming factor(s). Consistent with this, recent studies demonstrated that Not1 [80] and Not5 [81] form novel biomolecular condensates to enable elaborate translational regulation, while other subunits of the Ccr4-Not complex such as Not2, Ccr4, and Pop2, are components of the processing bodies [82]. Furthermore, the liquid-like state of the processing bodies was maintained in an Hsp104-dependent manner [83]. To confirm whether the Ccr4-Not complex is a true promoter reprograming factor, the effect of gene disruption of this complex on Taf1/ TFIID dependence of the PGK1 promoter would need to be evaluated in a strain generated similarly to the #3 strain in the present study. In any case, identification of promoter reprograming factor(s) would provide important insights into understanding how prions or biomolecular condensates are involved in TFIID-and SAGA-mediated gene transcription.  1-4, 9-10, 13-14, 17-18, 21-22, 25-26, and 29-30) or the VTC1 # reporter (lanes 5-8, 11-12, 15-16, 19-20, 23-24, 27-28, and 31-32) driven by the PGK1 promoter in which the TATA box was intact (lanes 1, 3, 5, 7, and 9-20) or substituted with the GAGA sequence (lanes 2, 4, 6, 8, and 21-32), as indicated below the bottom panel. The strains used in lanes 1-8 were the same as those used in lanes 1, 2, 3, 4, 7, 8, 9, and 10 of Fig 2B, respectively, as indicated. The other strains used in lanes 9-20 and 21-32 were haploid progenitors obtained by tetrad dissection of the mother #3/#2 diploid strains used in lanes 5/9 and 6/10 of Fig 2C, 1-6) or the VTC1 # reporter (lanes 7-12) driven by the PGK1 promoter in which the TATA was intact (odd-numbered lanes) or substituted with the GAGA sequence (even-numbered lanes), as indicated below the bottom panel. These strains are the same as those used in Fig 2B. Cultivation and data presentation were conducted as described in Fig 1A, except that the media used here contained 3 mM Gdn-HCl. (B) RT-qPCR analysis to measure mRNA levels of PGK1 (top panel) or VTC1/VTC1 # (bottom panel) in the ten diploid strains carrying both of the VTC1 and VTC1 # reporters driven by the PGK1 promoter in which the TATA box was intact (lanes 1/7, 3/9, 3/11, 5/9, and 5/11) or substituted with the GAGA sequence (lanes 2/8, 4/10, 4/12, 6/10, and 6/12), as indicated below the bottom panel. The strains are the same as those used in Fig 2C. Cultivation and data presentation were conducted as described in Fig 1A,